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Cardiovascular Research 2002 53(2):414-422; doi:10.1016/S0008-6363(01)00488-6
© 2002 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

p38 MAPK inhibition reduces myocardial reperfusion injury via inhibition of endothelial adhesion molecule expression and blockade of PMN accumulation

Feng Gaoa,1, Tian-Li Yueb,1, Dong-Wei Shia, Theodore A Christophera, Bernard L Lopeza, Eliot H Ohlsteinb, Frank C Baroneb and Xin L Maa,*

aDivision of Emergency Medicine, Jefferson Medical College, Thomas Jefferson University, 1020 Sansom Street, Philadelphia, PA 19107-5004, USA
bDepartment of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA, USA

* Corresponding author. Tel.: +1-215-955-4994; fax: +1-215-923-6225 xin.ma{at}mail.tju.edu

Received 21 June 2001; accepted 28 September 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: In vitro evidence suggests that the p38 mitogen-activated protein kinase (p38 MAPK) plays a crucial role in PMN activation and inflammatory cytokine production. However, the effect of p38 MAPK on myocardial reperfusion injury, a pathologic condition involving a typical inflammatory response, has not been fully examined. In the present study, we investigated the effect of SB 239063, a specific p38 MAPK inhibitor, on myocardial injury in a murine ischemia/reperfusion (I/R) model and elucidated the mechanism by which p38 MAPK inhibitor may exert its protective effect against I/R injury. Methods and results: I/R resulted in a significant myocardial injury (myocardial infarct 45±2.9%) and marked PMN accumulation (myeloperoxidase activity 1.03±0.16 U/100 g tissue). Administration of SB 239063 significantly inhibited the myocardial inflammatory response as evidenced by reduced PMN accumulation in I/R myocardial tissue (0.62±0.008 U/100 g tissue, P<0.01 vs. vehicle), and markedly attenuated myocardial reperfusion injury (myocardial infarct size: 28±2.4%, P<0.01 vs. vehicle). Moreover, treatment with SB 239063 significantly attenuated I/R-induced P-selectin and ICAM-1 upregulation (13.8±2.7 vs. 23.9±3.1%, and 29.4±1.6 vs. 56.3±4.8%, respectively P<0.01). In addition, pre-treatment with R15.7, a monoclonal antibody against CD 18 adhesion molecule on PMN surface that virtually abolished PMN accumulation in ischemic-reperfused myocardial tissue, significantly, but not completely, blocked the cardioprotection exerted by SB 239063. Conclusion: These results demonstrated for the first time that p38 MAPK activation plays a significant role in adhesion molecule upregulation on ischemia-reperfused endothelial cells and is an important signaling step in the pathogenesis of PMN-mediated tissue injury.

KEYWORDS Infection/inflammation; Ischemia; Reperfusion; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Early reperfusion after coronary obstruction is the most effective means of limiting ischemic myocardial injury. However, abundant evidence suggests that reperfusion may cause additional cell death (reperfusion injury). The process of myocardial ischemia/reperfusion (MI/R) injury involves components of a typical inflammatory reaction in which polymorphonuclear leukocytes (PMNs) play an important role. Experimental interventions aimed at reducing PMN accumulation (PMN depletion, pharmacological suppression of PMN activation, or administration of monoclonal antibodies against PMN or endothelial adhesion molecules) have been proven to exert significant protective effects on ischemia–reperfusion-induced myocardial damage [1–4].

Adhesion of PMNs to endothelial cells (ECs) is an early and requisite event in the inflammatory response. Current evidence indicates that adhesion of PMNs to ECs involves several steps and requires multiple adhesion molecules on the surface of both cell types [5]. The initial interaction between PMNs and ECs is mediated by L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) located on the PMN surface and P-selectin and E-selectin expressed on the EC surface. This selectin-mediated interaction results in the rolling of PMNs along the vessel wall and upregulation of β2-integrin adhesion molecules on the PMN surface [6]. Upregulated CD11/CD18 adhesion molecules then bind to their counterreceptors on ECs, primarily intercellular adhesion molecule-1 (ICAM-1), resulting in their firm adhesion and transmigration through the vascular wall. Although clear evidence exists demonstrating that adhesion molecule expression by coronary endothelial cells is markedly upregulated following myocardial ischemia and reperfusion [7], the signal transduction pathway leading to this response is poorly understood.

The mitogen-activated protein kinases (MAPKs) are a family of serine–threonine protein kinases that are activated in response to a variety of stimuli, such as growth factors and cellular stresses [8]. The three major MAPK signaling pathways that have been identified in mammalian cells involve extracellular signal-regulated protein kinases (ERK1/2), p38 MAPK and c-Jun NH2-terminal protein kinases (JNKs)/stress-activated protein kinases (SAPKs). Substantial evidence suggests that p38 MAPK plays a crucial role in the PMN response to a variety of stimuli. Inhibition of p38 MAPK in vitro reduces CD11/CD18 expression, PMN adhesion, and free radical generation [9–16]. However, to date, the role of p38 MAPK in adhesion molecule expression on the endothelial surface remains largely unexplored.

Therefore, the goals of the current study were: (1) to determine whether p38 MAPK inhibition may exert its cardioprotective effects against reperfusion injury via inhibition of the myocardial inflammatory response; and if so, (2) to investigate the mechanism by which p38 MAPK inhibition may inhibit PMN/EC interaction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Experimental protocol
The experiments were performed in adherence to National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Thomas Jefferson University Committee on Animal Care. Male Sprague–Dawley rats (200–250 g) were randomly divided into the following groups: sham MI/R rats (n=28), MI/R rats receiving vehicle (n=29), MI/R rats receiving SB 239063 (trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl) imidazole) (n=29), MI/R rats receiving R15.7 (a monoclonal antibody against CD18, 1 mg/kg, i.v., n=12), and MI/R rats receiving R15.7 and SB239063 (n=12). SB 239063 is a second-generation p38 MAPK inhibitor that has been demonstrated to be extremely selective for p38 MAPK (IC50 for inhibition of p38 MAPK: 0.044 µM; for MEK1/2, ERK1/2 and SAPK: >10 µM) [17]. SB 239063 was administered at a dose of 30 mg/kg, p.o., 20 min before the induction of myocardial ischemia. This is a dose regimen that completely blocked p38 MAPK activation, decreased TNF{alpha} production, and reduced brain injury and neurological deficits in cerebral focal ischemia [17]. Rats were anesthetized with pentobarbital sodium (35 mg/kg, i.p.) and the trachea was cannulated with PE-240 tubing to insure a patent airway. A midline thoracotomy was performed, the heart was exposed, and myocardial ischemia was produced by placing a 4-0 silk slipknot around the left coronary artery, approximately 2–3 mm from its origin. Ischemia was maintained for 30 min, at which time the slipknot was released, initiating reperfusion. Sham operated control rats (Sham MI/R) underwent the same surgical procedures except that the suture that was passed under the left coronary artery was not tied. After 10 min (for p38 MAPK activity), 20 min (for P-selectin immunolocalization), or 24 h of reperfusion (for ICAM-1 immunolocalization, myocardial myeloperoxidase and myocardial infarct size), the hearts were excised and processed according to the procedures described below for evaluation of myocardial infarct size, myocardial myeloperoxidase (MPO) activity, and adhesion molecule expression.

2.2 Determination of myocardial infarct size
At the end of the 24-h reperfusion period, the ligature around the coronary artery was retied and 1 ml of 2% Evans blue dye was injected into the left ventricular cavity. The dye was circulated and uniformly distributed except in that portion of the heart previously perfused by the occluded coronary artery (area-at-risk, AAR). The heart was quickly excised, frozen at –20°C, and sliced into 1-mm-thick sections perpendicular to the long axis of the heart using a heart slice chamber. Slices were incubated individually using a 24-well culture plate in 1% TTC in phosphate buffer at pH 7.4 at 37°C for 10 min, and photographed with a digital camera (1600x1200 dpi). Evan's blue stained area (area-not-at-risk, ANAR), TTC stained area (red staining, ischemic but viable tissue), and TTC staining negative area (infarct myocardium) were digitally measured using SigmaScan. The myocardial infarct size was expressed as a percentage of infarct area over total AAR.

2.3 Determination of myocardial myeloperoxidase activity
Myeloperoxidase, an enzyme occurring virtually exclusively in PMNs, was determined in cardiac tissue as described previously [18]. In brief, ischemic/reperfused cardiac tissue was homogenized in 50 mmol/l potassium phosphate buffer at pH 6 containing 0.5% hexadecyltrimethyl ammonium bromide (HTAB). The homogenates were centrifuged for 30 min at 12 500xg and 4°C. The supernatants were then collected and reacted with 0.167 mg/ml o-dianisodine dihydrochloride and 0.0005% H2O2 in 50 mmol/l phosphate buffer at pH 6. The measurement was performed at 460 nm at 25°C using a microplate reader (SpectraMAX Plus, Molecular Devices, Sunnyvale, CA) in duplicate. One unit of MPO activity was defined as that quantity of enzyme hydrolyzing 1 mmol of peroxide per min at 25°C.

2.4 Immunohistochemical localization of P-selectin
After 30 min of ischemia and 20 min of reperfusion, the hearts were removed and the aorta was cannulated and perfused with KH buffer for 3 min at 50 mmHg. After the heart was cleared of blood, perfusion was switched to ice-cold 4% paraformaldehyde in PBS for 5 min to perfusion fix the hearts. Four full thickness slices of the ischemic and non-ischemic left ventricular wall were cut and immersed in 4% paraformaldehyde for 24 h. Afterwards, they were processed (Leica TP1050 infiltration processor), embedded in paraffin (Leica EG 1140H Bedienhinweise) and sectioned at 5-µm (Leica RM2035 Microtome). The tissue sections were placed on Vectabond-coated slides (Vector Laboratories, Burlingame, CA). Immunohistochemical localization of P-selectin was accomplished using a rabbit anti-human P-selectin polyclonal antibody (09361A, Pharmingen, San Diego, CA) by the avidin–biotin immunoperoxidase technique (Vectastain ABC reagent; Vector Laboratories). Positive staining was defined as a venule displaying brown reaction product on >50% of the circumference of its endothelium as previously described [19,20]. Ten sections from each heart and 20 venules per tissue section were examined, and the percentage of positive staining venules was then calculated.

2.5 Immunohistochemical localization of ICAM-1
It has been previously reported by several investigators that ICAM-1 immunohistochemical localization cannot be performed using paraformaldehyde fixed tissue [21,22]. Frozen sections were thus used and stained according to the manufacturer's (Pharmingen) instructions. In brief, after 30 min of ischemia and 24 h of reperfusion, the hearts were removed and washed with ice-cold PBS. Four full thickness slices of the ischemic and non-ischemic left ventricular wall were immediately embedded in OCT-compound and frozen in liquid nitrogen. Cryostat sections of 6 µm thickness were cut, fixed in cold acetone and air-dried. The sections were pretreated for 10 min with 0.03% H2O2 in PBS to quench endogenous peroxidase and blocked with 5% normal serum. This was followed by incubation with primary antibody (10 µg/ml mouse anti-rat ICAM-1 monoclonal antibody, 22491D, Pharmingen) overnight at 4°C in humidified chambers. After washing, the sections were incubated with biotinylated secondary antibody at room temperature for 1 h. Antibody-binding sites were labeled and measured in the same manner as that described above for P-selectin immunohistochemical localization.

2.6 p38 MAP kinase activity assay
The p38 MAP kinase activity assay was performed using a p38 MAPK assay kit (Cell Signaling Technology) according to the manufacturer's instructions. In brief, ischemic/reperfused (30 min/10 min) heart tissue (20–25 mg) was homogenized in 0.5 ml ice-cold cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, 1 mM PMSF). The lysates were then sonicated on ice and centrifuged at 15 000xg for 10 min at 4°C. Immunoprecipitation was performed by adding 20 µl of resuspended immobilized monoclonal antibody against phospho-p38 MAP kinase (Thr180/Tyr182) to 200 µl cell lysate containing 500 µg protein. The mixture was incubated with gentle rocking overnight at 4°C. After centrifuging at 10 000xg at 4°C for 2 min, the pellets were washed twice with lysis buffer and twice with kinase buffer (25 mM Tris, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mN MgCl2). The kinase reactions were carried out in the presence of 200 µM ATP and 2 µg ATF-2 fusion protein at 30°C for 30 min. After incubation, the samples were separated by SDS–PAGE, and ATF-2 phosphorylation was measured by Western immunoblotting using a monoclonal antibody against phosphorylated ATF-2 followed by an enhanced chemiluminescent detection.

2.7 Statistical analysis
All values in the text and figures are presented as means±standard errors (S.E.) of n independent experiments. All data were subjected to analysis of variance (ANOVA) followed by the Scheffe's correction for post-hoc t-test comparison. Probabilities of 0.05 or less were considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Ischemia/reperfusion activation of p38 MAPK and its blockade by SB 239063
Effect of in vivo myocardial ischemia and reperfusion on p38 MAPK activity and its inhibition by SB 239063 was determined using a p38 MAPK assay kit. The data show that a transient (<30 min), moderate (two- to threefold) increase in p38 MAPK activity was observed in the hearts subjected to ischemia without reperfusion (data not shown). However, when ischemic myocardial tissue was reperfused, p38 MAPK was markedly reactivated. A 4.5-fold increase in p38 MAPK activity was observed at 10 min after reperfusion (Fig. 1) and its activity gradually returned to control level thereafter. Treatment with SB 239063 at the dose regimen selected virtually abolished p38 MAPK activation and brought p38 MAPK activity to a level that was not statistically different from those seen in hearts subjected to sham MI/R (Fig. 1). These results clearly demonstrated that p38 MAPK was activated by ischemia and reperfusion, and its activation can be inhibited by SB 239063 at the dose regimen selected in the present experiment.


Figure 1
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  		Fig. 1 p38 MAPK activities in ischemic/reperfused (30 min/10 min) myocardial tissue from sham MI, MI treated with vehicle, or MI treated with SB 239063. The p38 MAP kinase activity assay was performed using a p38 MAPK assay kit with ATF-2 as substrate (see details in the Methods section). Insert: representative blots; bar heights represent mean values of densitometric evaluation and brackets indicate S.E. n=6 in each group. **P<0.01 vs. vehicle-treated ischemia-reperfused hearts.

 
3.2 Effect of p38 MAPK inhibition on myocardial infarct size after ischemia/reperfusion
To determine whether inhibition of p38 MAPK may exert significant cardioprotection against reperfusion injury, we investigated the effect of SB 239063 treatment on myocardial infarct size after 30 min of ischemia and 24 h of reperfusion. As illustrated in Fig. 2, ischemia followed by reperfusion resulted in a significant myocardial infarct, which was significantly attenuated with SB 239063 treatment.


Figure 2
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  		Fig. 2 Myocardial infarct size in the different treatment groups. Upper portion: representative photomicrographs (x25 magnification) of heart sections obtained from rats subjected to sham myocardial ischemia, ischemia/reperfusion treated with vehicle, or ischemia/reperfusion treated with SB 239063. Black stained portion=non-ischemic, normal region; Red stained portion=ischemic/reperfused, but not infarcted region; Negative stained portion=ischemic/reperfused, infarcted region. Lower portion: Bar illustration of myocardial infarct size expressed as percent of total ischemic-reperfused area (area-at-risk). Bar heights represent mean values and brackets indicate S.E. At least 12 rats were studied in each group. **P<0.01 versus vehicle-treated ischemia-reperfused hearts; #P<0.05 versus R15.7 treatment alone.

 
3.3 Effect of p38 MAPK inhibition on PMN accumulation in ischemic/reperfused cardiac tissue
To determine whether p38 MAPK inhibition may exert its cardioprotective effects by inhibiting PMN accumulation and thus reducing PMN-induced myocardial injury, we measured MPO activity, an index that has been shown to correlate closely with PMN accumulation in the heart [23]. As summarized in Fig. 3, 30 min of ischemia and 24 h of reperfusion resulted in a sixfold increase in MPO activity, indicating that there was significant PMN accumulation in ischemic/reperfused cardiac tissue. This increased MPO activity in ischemic/reperfused tissue was significantly attenuated by treatment with SB 239063. This result demonstrated that blockade of p38 MAPK significantly decreased PMN accumulation in ischemic/reperfused cardiac tissue, suggesting that p38 MAPK inhibition exerted its protective effects partially through inhibiting PMN/EC interaction and subsequent inflammatory injury to the myocardial tissue.


Figure 3
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  		Fig. 3 MPO activity in ischemia/reperfused myocardial tissue (30 min/24 h, see details in the Methods section). Bar heights represent mean values and brackets indicate S.E. At least 12 rats were studied in each group. **P<0.01 versus vehicle-treated ischemia-reperfused hearts. SB=SB 239063; R15.7=monoclonal antibody against CD18 adhesion molecule on PMN surface.

 
3.4 Effect of p38 MAPK inhibition on EC surface expression of P-selectin and ICAM-1 following MI/R
To further explore the mechanisms by which p38 MAPK inhibition exerts its protection against PMN-induced myocardial injury following reperfusion, we studied the effect of SB 239063 on endothelial surface expression of P-selectin, an early-response adhesion molecule mediating PMN rolling, and ICAM-1, a critical molecule required for PMN firm adhesion and transmigration. The percentage of coronary venules staining positively for P-selectin and ICAM-1 in sham MI/R rats was low (Fig. 4). Thirty minutes of ischemia followed by 20 min of reperfusion resulted in a significant increase in the percentage of venules staining positively for P-selectin in untreated ischemic/reperfused rats (Fig. 4). Treatment with SB 239063 at a dose that blocked p38 MAPK activation after ischemia and reperfusion reduced positive staining (13.8±2.7%). This represented a 51% inhibition of P-selectin upregulation in rats receiving only vehicle. In rats exposed to 30 min of ischemia and 24 h of reperfusion and receiving only vehicle, ICAM-1 positive staining increased 4.5-fold compared to staining in sham MI/R rat venules (Fig. 4). This increase in percentage of venules staining positively for ICAM-1 was markedly reduced in rats treated with SB 239063 (Fig. 4). A previous study has demonstrated that endothelial surface ICAM-1 expression is significantly upregulated as early as 4.5 h after reperfusion, and reached the maximal level at 24 h after reperfusion [7]. To determine whether treatment with SB 239063 also attenuates the early phase of ICAM-1 upregulation, another 10 animals (five rats in each group) were treated with either vehicle or SB 239063 and sacrificed at 4.5 h after reperfusion. The data demonstrated that treatment with SB 239063 also significantly reduced ICAM-1 upregulation at this time point (ICAM-1 positive venules: 19.8±2.6 vs. 42.3±3.9% in vehicle-treated animals, P<0.01).


Figure 4
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  		Fig. 4 Number of peroxidase stained venules expressed as percent of the total number of venules [(stained venules/total venules)x100]. The ratio of stained venules to the total number of vessels was calculated for each rat heart exposed to 30 min of ischemia and 20 min (P-selectin) or 24 h (ICAM-1) of reperfusion. The data are presented as means±S.E.M. for 6–8 rats in each group. **P<0.01 versus vehicle-treated ischemia-reperfused hearts. Insert: Photomicrograph of heart tissue incubated with anti-ICAM-1 monoclonal antibody and labeled with peroxidase substrate solution. (a) A negatively stained venule from a heart subjected to sham myocardial ischemia/reperfusion; (b) a positively stained venule from a heart subjected to 30 min of ischemia and 24 h of reperfusion. The red line represents only the stained portion of the endothelial perimeter.

 
3.5 Contribution of anti-inflammatory effect of SB 239063 on its overall cardioprotection after in vivo myocardial ischemia and reperfusion
Several previous studies, including one from our laboratory, demonstrated that inhibition of p38 MAPK exerts significant cardioprotection in isolated hearts perfused with crystalloid solution without PMNs, indicating that activation of p38 MAPK can also induce myocardial injury via mechanisms that are independent of PMNs [24–27]. To determine the relative contribution of PMN-dependent and PMN-independent components of p38 MAPK activation on reperfusion injury, another two groups of animals were studied. The first group of rats were treated with R15.7 (1 mg/kg, i.v., 20 min before coronary artery occlusion), a monoclonal antibody against CD18 that has been demonstrated to block PMN adhesion and reduce myocardial reperfusion injury in rat [28]. The second group of rats were treated with a combination of R15.7 and SB 239063. Consistent with previously published results [28], treatment with R15.7 virtually abolished the increase in myocardial MPO activity, and markedly reduced infarct size (Figs. 2 and 3Go). Most interestingly, although treatment with SB 239063 in addition to R15.7 did not have a significant effect on myocardial MPO activity, this treatment further reduced myocardial infarct size to a level that was significantly smaller than those hearts treated with R15.7 alone (Fig. 2). These results suggest that although the anti-adhesion and anti-inflammatory effects of p38 MAPK inhibition is largely responsible for observed cardioprotection in this in vivo myocardial ischemia and reperfusion model, there is an additional PMN-independent component that contributes to its overall cardioprotection against reperfusion injury.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrated that blockade of p38 MAPK: (1) reduced ischemia–reperfusion-induced myocardial infarct; (2) attenuated PMN accumulation in ischemic-reperfused myocardial tissue; and (3) inhibited P-selectin and ICAM-1 surface expression in ischemic-reperfused coronary endothelial cells. To our knowledge, this is the first study that has: (1) investigated the role of p38 MAPK in signal transduction leading to adhesion molecule expression in ischemic-reperfused coronary endothelial cells in vivo and (2) demonstrated that p38 MAPK inhibition may exert significant cardioprotection by inhibiting PMN accumulation and the subsequent myocardial inflammatory injury.

Considerable evidence suggests that endothelial surface expressed P-selectin binding to its PMN ligands may not only result in PMN rolling along the endothelial surface but may also initiate a signal within PMNs that leads to β2-integrin upregulation, thus resulting in CD11/CD18-dependent adhesion to ICAM-1 [29,30,6]. Several recent studies have demonstrated that p38 MAPK plays a key role in this selectin–β2-integrin cross-talk process. Schnyder et al. [9] reported that inhibition of p38 MAPK reduced CD11b surface expression on fMLP-stimulated PMNs and impaired PMN activation and killing of Staphylococcus aureus. Detmers et al. [13] demonstrated that SB203580, a specific p38 MAPK inhibitor, significantly inhibited TNF{alpha}-activated PMN adhesion to fibrinogen-coated plates and reduced H2O2 production by adhered PMNs. Yaffe et al. [12] and Partrick et al. [14] reported that p38 MAPK activation is required for priming of the PMN respiratory burst in response to TNF{alpha}. In addition, Johnson et al. [11] demonstrated that two members of the MAPK family, p38 MAPK and ERK, are activated in fMLP-stimulated PMNs. However, the roles of p38 MAPK and ERK in PMN cytotoxicity were opposite. Activation of p38 MAPK increased superoxide release and enhanced PMN cytotoxicity, whereas ERK activation reduced elastase release and decreased PMN cytotoxicity. Moreover, three groups of investigators have recently demonstrated that hypertonic saline inhibits CD11/CD18 upregulation in agonist-stimulated PMNs in a p38 MAPK-dependent fashion [16,15,10]. Taken together, these results demonstrate that p38 MAPK plays a crucial role in signal transduction in PMNs following their binding with P-selectin on the endothelial surface. However, the role of p38 MAPK in P-selectin surface expression by endothelial cells has not been previously elucidated.

In the present study, we have provided direct evidence that p38 MAPK also plays a crucial role in the signal transduction leading to P-selectin translocation in endothelial cells. We have demonstrated for the first time that treatment with SB 239063, a highly specific p38 MAPK inhibitor, markedly attenuated P-selectin expression in coronary endothelial cells exposed to 30 min of ischemia and 20 min of reperfusion. These results suggest that p38 MAPK may increase P-selectin-dependent PMN–EC interactions in at least two ways. First, p38 MAPK activation leads to P-selectin translocation in ischemic/reperfused coronary endothelial cells, thus facilitating PMN rolling along the endothelial surface. Second, p38 MAPK participates in selectin–β2-integrin cross-talk and upregulates CD11/CD18 expression on PMN surfaces, thus increasing integrin/ICAM-1-dependent PMN adhesion.

Another interesting finding of the present study is that blockade of p38 MAPK activation inhibited ICAM-1 expression in ischemic/reperfused coronary endothelial cells. Although existing data uniformly support the conclusion that p38 MAPK is involved in upregulation of adhesion molecule expression on PMN surfaces, the evidence that p38 MAPK regulates adhesion molecule expression on endothelial cells is limited. Reported data are controversial and all previous studies were performed in cultured cells and stimulated with cytokines in vitro. In human umbilical vein endothelial cells exposed to TNF{alpha}, Pietersma et al. [31] reported that p38 MAPK is involved with posttranscriptional activation of vascular cell adhesion molecule-1 (VCAM-1) but not ICAM-1. In contrast, Tamura et al. [32] have demonstrated that signaling through p38 MAPK contributes to LPS and TNF{alpha} stimulated ICAM-1 surface expression in human pulmonary microvascular endothelial cells. Consistent with this finding, Kacimi et al. recently [33] reported that p38 MAPK plays a significant role in ICAM-1 upregulation in cultured cardiac cells after cytokine stimulation. Our present study provides the first in vivo evidence that p38 MAPK is a key component in signal transduction leading to ICAM-1 upregulation in coronary endothelial cells exposed to ischemia and reperfusion. Considerable evidence exists that myocardial ischemia followed by reperfusion increases cytokine production, and this increased cytokine production in turn results in upregulation of endothelial surface adhesion molecules such as ICAM-1 and E-selectin [34]. On the other hand, Barone et al. recently reported that treatment with SB 239063 markedly reduced plasma TNF{alpha} concentration following in vivo LPS injection [17]. Taken together, it is conceivable that treatment with SB 239063 in rats subjected to myocardial ischemia and reperfusion may reduce cytokine production, thus attenuating ICAM-1 expression.

A previous study has demonstrated that in cultured endothelial cells stimulated with TNF{alpha}, blockade of p38 MAPK resulted in a 40% reduction in ICAM-1 upregulation [9]. However, the physiologic significance of this partial reduction of adhesion molecules remains unclear. Specifically, a concern has been expressed as to whether or not this incomplete down-regulation of ICAM-1 expression is sufficient to cause a significant reduction in PMN–EC interaction. In the present study, we have demonstrated that administration of SB 239063 significantly reduced MPO activity, a reliable index of PMN accumulation in ischemic-reperfused cardiac tissue. This result provides direct evidence that inhibition of adhesion molecule expression by blockade of p38 MAPK signaling indeed causes a significant reduction in PMN–EC interaction. To further determine whether inhibition of adhesion molecule expression and reduction of PMN accumulation may reduce myocardial reperfusion injury, we investigated the effects of SB 239063 on myocardial infarct after ischemia and reperfusion. Our data clearly demonstrate that inhibition of p38 MAPK markedly attenuated ischemia–reperfusion-induced myocardial injury as evidenced by reduced myocardial infarct size in SB 239063-treated animals.

It needs to be stressed that the cardioprotection elicited by p38 MAPK inhibition is likely to be multifactorial and involves PMN-dependent as well as PMN-independent mechanisms. The relative contribution of each component of p38 MAPK inhibition on its overall cardioprotection may differ substantially depending on the experimental models used. In the isolated perfused heart model where myocardial reperfusion injury is caused by mechanisms other than PMN accumulation and inflammatory response, inhibition of p38 MAPK may attenuate reperfusion injury by blocking intracellular signal transduction pathways leading to myocardial apoptosis [24–27]. In contrast, in an in vivo regional myocardial ischemia and reperfusion model where PMN accumulation and inflammatory response is largely responsible for myocardial reperfusion injury [3,4], blockade of PMN adhesion to the endothelial surface appears to be the primary mechanism by which p38 MAPK inhibition exerts its cardioprotection against reperfusion injury. In this connection, our present experiment provides direct evidence that anti-adhesion and anti-inflammatory effects of p38 MAPK blockade is largely, but not solely, responsible for observed cardioprotection in the in vivo myocardial ischemia and reperfusion model.

In summary, the present study demonstrated that p38 MAPK activation is an important signaling step in the pathogenesis of PMN-mediated tissue injury. In addition to its reported role in P-selectin–β2-integrins cross-talk in PMN activation, p38 MAPK appears to play a crucial role in the surface expression of P-selectin as well as ICAM-1 on coronary endothelial cells exposed to ischemia and reperfusion. Inhibition of p38 MAPK activity may therefore attenuate surface expression of the adhesion molecules required for both PMN rolling (P-selectin) and firm adhesion (ICAM-1), thus significantly reducing PMN accumulation in ischemic reperfused myocardial tissue and attenuating PMN-initiated tissue injury. Accordingly, inhibition of p38 MAPK activity may be a therapeutic target in the prevention of PMN-mediated tissue destruction seen with hyperinflammatory disorders such as myocardial ischemia and reperfusion.

Time for primary review 26 days.


   Acknowledgements
 
This research was supported in part by NIH grant HL-63828, NSFC grants 39925013, 39970807 (X.L. Ma), and NSFC grant 39970302 (F. Gao). We gratefully acknowledge the generous supply of R15.7 monoclonal antibody received from Dr Robert Rothlein of Boehringer Ingelheim Pharmaceuticals in Ridgefield, CT.


   Notes
 
1 The first two authors made an equal contribution to this study. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Ma X.L., Tsao P.S., Lefer A.M. Antibody to CD-18 exerts endothelial and cardiac protective effects in myocardial ischemia and reperfusion. J Clin Invest (1991) 88:1237–1243.[Web of Science][Medline]
  2. Ma X.L., Lefer D.J., Lefer A.M., Rothlein R. Coronary endothelial and cardiac protective effects of a monoclonal antibody to intercellular adhesion molecule-1 in myocardial ischemia and reperfusion. Circulation (1992) 86:937–946.[Abstract/Free Full Text]
  3. Lefer A.M., Ma X.L., Weyrich A., Lefer D.J. Endothelial dysfunction and PMN adherence as critical events in the development of reperfusion injury. Agents Actions Suppl (1993) 41:127–135.[Medline]
  4. Chandrasekar B., Smith J.B., Freeman G.L. Ischemia-reperfusion of rat myocardium activates nuclear factor-KappaB and induces PMN infiltration via lipopolysaccharide-induced CXC chemokine. Circulation (2001) 103:2296–2302.[Abstract/Free Full Text]
  5. Zimmerman G.A., McIntyre T.M., Prescott S.M. Adhesion and signaling in vascular cell–cell interactions. J Clin Invest (1996) 98:1699–1702.[Web of Science][Medline]
  6. Cerletti C., Evangelista V., de Gaetano G. P-selectin–beta 2-integrin cross-talk: a molecular mechanism for polymorphonuclear leukocyte recruitment at the site of vascular damage. Thromb Haemost (1999) 82:787–793.[Web of Science][Medline]
  7. Weyrich A.S., Buerke M., Albertine K.H., Lefer A.M. Time course of coronary vascular endothelial adhesion molecule expression during reperfusion of the ischemic feline myocardium. J Leukoc Biol (1995) 57:45–55.[Abstract]
  8. Waskiewicz A.J., Cooper J.A. Mitogen and stress response pathways: map kinase cascades and phosphatase regulation in mammals and yeast. Curr Opin Cell Biol (1995) 7:798–805.[CrossRef][Web of Science][Medline]
  9. Schnyder B., Meunier P.C., Car B.D. Inhibition of kinases impairs PMN activation and killing of Staphylococcus aureus. Biochem J (1998) 331:489–495.[Web of Science][Medline]
  10. Ciesla D.J., Moore E.E., Gonzalez R.J., Biffl W.L., Silliman C.C. Hypertonic saline inhibits PMN (PMN) priming via attenuation of p38 MAPK signaling. Shock (2000) 14:265–269.[Web of Science][Medline]
  11. Johnson J.L., Moore E.E., Partrick D.A., Tamura D.Y., Zallen G., Elzi D.J., et al. Extracellular signal-related kinase 1/2 and p38 mitogen-activated protein kinase pathways serve opposite roles in PMN cytotoxicity. Arch Surg (1999) 134:1074–1078.[Abstract/Free Full Text]
  12. Yaffe M.B., Xu J., Burke P.A., Forse R.A., Brown G.E. Priming of the PMN respiratory burst is species-dependent and involves MAP kinase activation. Surgery (1999) 126:248–254.[Web of Science][Medline]
  13. Detmers P.A., Zhou D., Polizzi E., Thieringer R., Hanlon W.A., Vaidya S., et al. Role of stress-activated mitogen-activated protein kinase (p38) in beta 2-integrin-dependent PMN adhesion and the adhesion-dependent oxidative burst. J Immunol (1998) 161:1921–1929.[Abstract/Free Full Text]
  14. Partrick D.A., Moore E.E., Offner P.J., Meldrum D.R., Tamura D.Y., Johnson J.L., et al. Maximal human PMN priming for superoxide production and elastase release requires p38 mitogen-activated protein kinase activation. Arch Surg (2000) 135:219–225.[Abstract/Free Full Text]
  15. Rizoli S.B., Kapus A., Parodo J., Rotstein O.D. Hypertonicity prevents lipopolysaccharide-stimulated CD11b/CD18 expression in human PMNs in vitro: role for p38 inhibition. J Trauma (1999) 46:794–798.[Web of Science][Medline]
  16. Junger W.G., Hoyt D.B., Davis R.E., Herdon-Remelius C., Namiki S., Junger H., et al. Hypertonicity regulates the function of human PMNs by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38. J Clin Invest (1998) 101:2768–2779.[Web of Science][Medline]
  17. Barone F.C., Irving E.A., Ray A.M., Lee J.C., Kassis S., Kumar S., et al. SB 239063, a second-generation p38 mitogen-activated protein kinase inhibitor, reduces brain injury and neurological deficits in cerebral focal ischemia. J Pharmacol Exp Ther (2001) 296:312–321.[Abstract/Free Full Text]
  18. Ma X.L., Yue T.L., Lopez B.L., Barone F.C., Christopher T.A., Ruffolo R.R. Jr., et al. Carvedilol, a new beta-adrenoreceptor blocker and free radical scavenger, attenuates myocardial ischemia–reperfusion injury in hypercholesterolemic rabbits. J Pharmacol Exp Ther (1996) 277:128–136.[Abstract/Free Full Text]
  19. Lefer A.M., Campbell B., Scalia R., Lefer D.J. Synergism between platelets and PMNs in provoking cardiac dysfunction after ischemia and reperfusion — Role of selectins. Circulation (1998) 98:1322–1328.[Abstract/Free Full Text]
  20. Nossuli T.O., Hayward R., Jensen D., Scalia R., Lefer A.M. Mechanisms of cardioprotection by peroxynitrite in myocardial ischemia and reperfusion injury. Am J Physiol Heart Circ Physiol (1998) 275:H509–H519.[Abstract/Free Full Text]
  21. Hawkins H.K., Entman M.L., Zhu J.Y., Youker K.A., Berens K., Doré M., et al. Acute inflammatory reaction after myocardial ischemic injury and reperfusion — Development and use of a PMN-specific antibody. Am J Pathol (1996) 148:1957–1969.[Abstract]
  22. Jaakkola K., Jalkanen S., Kaunismaki K., Vanttinen E., Saukko P., Alanen K., et al. Vascular adhesion protein-1, intercellular adhesion molecule-1 and P-selectin mediate leukocyte binding to ischemic heart in humans. J Am Coll Cardiol (2000) 36:122–129.[Abstract/Free Full Text]
  23. Mullane K.M., Kraemer R., Smith B. Myeloperoxidase activity as a quantitative assessment of PMN infiltration into ischemic myocardium. J Pharmacol Methods (1985) 14:157–167.[CrossRef][Web of Science][Medline]
  24. Ma X.L., Kumar S., Gao F., Louden C.S., Lopez B.L., Christopher T.A., et al. Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation (1999) 99:1685–1691.[Abstract/Free Full Text]
  25. Marais E., Genade S., Huisamen B., Strijdom J.G., Moolman J.A., Lochner A. Activation of p38 MAPK induced by a multi-cycle ischaemic preconditioning protocol is associated with attenuated p38 MAPK activity during sustained ischaemia and reperfusion. J Mol Cell Cardiol (2001) 33:769–778.[CrossRef][Web of Science][Medline]
  26. Gysembergh A., Simkhovich B.Z., Kloner R.A., Przyklenk K. p38 MAPK activity is not increased early during sustained coronary artery occlusion in preconditioned versus control rabbit heart. J Mol Cell Cardiol (2001) 33:681–690.[CrossRef][Web of Science][Medline]
  27. Schneider S., Chen W., Hou J., Steenbergen C., Murphy E. Inhibition of p38 MAPK {alpha}/{beta} reduces ischemic injury and does not block protective effects of preconditioning. Am J Physiol (2001) 280:H499–H508.[Web of Science]
  28. Lefer D.J., Shandelya S.M.L., Serrano C.V. Jr., Becker L.C., Kuppusamy P., Zweier J.L. Cardioprotective actions of a monoclonal antibody against CD-18 in myocardial ischemia–reperfusion injury. Circulation (1993) 88:1779–1787.[Abstract/Free Full Text]
  29. Blanks J.E., Moll T., Eytner R., Vestweber D. Stimulation of P-selectin glycoprotein ligand-1 on mouse PMNs activates beta 2-integrin mediated cell attachment to ICAM-1. Eur J Immunol (1998) 28:433–443.[CrossRef][Web of Science][Medline]
  30. Evangelista V., Manarini S., Rotondo S., Martelli N., Polischuk R., McGregor J.L., et al. Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18. Blood (1996) 88:4183–4194.[Abstract/Free Full Text]
  31. Pietersma A., Tilly B.C., Gaestel N., De Jong N., Lee J.C., Koster J.F., et al. P38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level. Biochem Biophys Res Commun (1997) 230:44–48.[CrossRef][Web of Science][Medline]
  32. Tamura D.Y., Moore E.E., Johnson J.L., Zallen G., Aiboshi J., Silliman C.C. p38 mitogen-activated protein kinase inhibition attenuates intercellular adhesion molecule-1 up-regulation on human pulmonary microvascular endothelial cells. Surgery (1998) 124:403–407.[Web of Science][Medline]
  33. Kacimi R., Karliner J.S., Koudssi F., Long C.S. Expression and regulation of adhesion molecules in cardiac cells by cytokines: response to acute hypoxia. Circ Res (1998) 82:576–586.[Abstract/Free Full Text]
  34. Frangogiannis N.G., Youker K.A., Rossen R.D., Gwechenberger M., Lindsey M.H., Mendoza L.H., et al. Cytokines and the microcirculation in ischemia and reperfusion. J Mol Cell Cardiol (1998) 30:2567–2576.[CrossRef][Web of Science][Medline]

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